1. Field of the Invention
The invention relates to the fields of industrial microbiology and alcohol production. Specifically, the invention relates suitable alcohol dehydrogenases for the production of lower alkyl alcohols via an engineered pathway in microorganisms. More specifically, the invention relates to suitable alcohol dehydrogenases for the production of butanol, particularly isobutanol, via an engineered pathway in microorganisms.
2. Background Art
Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a food grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase in the future.
Methods for the chemical synthesis of isobutanol are known, such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) and Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A: Chem. 220:215-220, 2004). These processes use starting materials derived from petrochemicals, are generally expensive, and are not environmentally friendly.
Isobutanol is produced biologically as a by-product of yeast fermentation. It is a component of “fusel oil” that forms as a result of the incomplete metabolism of amino acids by this group of fungi. Isobutanol is specifically produced from catabolism of L-valine. After the amine group of L-valine is harvested as a nitrogen source, the resulting α-keto acid is decarboxylated and reduced to isobutanol by enzymes of the so-called Ehrlich pathway (Dickinson et al., J. Biol. Chem. 273:25752-25756, 1998). Yields of fusel oil and/or its components achieved during beverage fermentation are typically low. For example, the concentration of isobutanol produced in beer fermentation is reported to be less than 16 parts per million (Garcia et al., Process Biochemistry 29:303-309, 1994). Addition of exogenous L-valine to the fermentation mixture increases the yield of isobutanol, as described by Dickinson et al., supra, wherein it is reported that a yield of isobutanol of 3 g/L is obtained by providing L-valine at a concentration of 20 g/L in the fermentation mixture. In addition, production of n-propanol, isobutanol and isoamylalcohol has been shown by calcium alginate immobilized cells of Zymomonas mobilis. A 10% glucose-containing medium supplemented with either L-Leu, L-Ile, L-Val, α-ketoisocaproic acid (α-KCA), α-ketobutyric acid (α-KBA) or α-ketoisovaleric acid (α-KVA) was used (Oaxaca, et al., Acta Biotechnol. 11:523-532, 1991). α-KCA increased isobutanol levels. The amino acids also gave corresponding alcohols, but to a lesser degree than the keto acids. An increase in the yield of C3-C5 alcohols from carbohydrates was shown when amino acids leucine, isoleucine, and/or valine were added to the growth medium as the nitrogen source (PCT Publ. No. WO 2005/040392).
Whereas the methods described above indicate the potential of isobutanol production via biological means, these methods are cost prohibitive for industrial scale isobutanol production.
For an efficient biosynthetic process, an optimal enzyme is required at the last step to rapidly convert isobutyraldehyde to isobutanol. Furthermore, an accumulation of isobutyraldehyde in the production host normally leads to undesirable cellular toxicity.
Alcohol dehydrogenases (ADHs) are a family of proteins comprising a large group of enzymes that catalyze the interconversion of aldehydes and alcohols (de Smidt et al., FEMS Yeast Res., 8:967-978, 2008), with varying specificities for different alcohols and aldehydes. There is a need to identify suitable ADH enzymes to catalyze the formation of product alcohols in recombinant microorganisms. There is also a need to identify a suitable ADH enzyme that would catalyze the formation of isobutanol at a high rate, with specific affinity for isobutyraldehyde as the substrate and in the presence of high levels of isobutanol.